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1.
目的研究肾草酸钙结石形成对肾组织bikunin基因和间α胰蛋白酶抑制物(IαI)蛋白表达的影响,探讨bikunin在尿结石形成中的意义。方法诱导实验性大鼠肾草酸钙结石模型,收集结石大鼠、正常大鼠、临床肾结石和非结石患者每组各8例的肾组织标本。采用免疫组化、逆转录聚合酶链反应(RT-PCR)和计算机图像分析技术分别检测所有大鼠和人肾组织中bikuninmRNA和IαI蛋白的表达水平。结果正常大鼠和非结石患者肾组织均存在bikuninmRNA和IαI蛋白的表达。肾草酸钙结石形成后,结石大鼠肾组织IαI蛋白的灰度值和bikuninmRNA的相对表达水平分别为198.43±15.17、0.70±0.14;肾结石患者肾组织IαI蛋白的灰度值和bikuninmRNA的相对表达水平分别为263.25±17.41、1.27±0.13,分别和对照组相比,均显著增加(P<0.05)。结论Bikunin作为构成IαI的轻链结构,在肾草酸钙结石形成后,bikuninmRNA的表达迅速增强,提示机体通过肾脏合成更多的bikunin来抑制肾草酸钙结石的形成。 相似文献
2.
目的研究中药泽泻活性成分对结石模型大鼠肾结石形成和bikunin表达的影响,以探讨泽泻防治草酸钙尿石症的作用机理.方法分离提取泽泻的化学活性成分,制作大鼠肾草酸钙结石模型.将Wistar大鼠随机分成对照组、结石模型组、泽泻组,采用半定量逆转录聚合酶链反应检测大鼠肾组织bikunin mRNA的表达水平、镜下观察肾组织草酸钙晶体分布,同时检测大鼠血生化、肾钙含量、24h尿钙和尿草酸的分泌量.结果①泽泻组大鼠肾组织草酸钙晶体分布、血生化等指标均明显低于结石组.②泽泻组和成石组大鼠肾组织bikunin的相对表达水平分别为(0.528±0.170,0.713±0.250)、肾钙含量分别为[(4.70±0.08)mg/g,(9.49±0.45)mg/g]、24h尿钙分泌量分别为[(37.23±1.84)μmol,(61.49±2.06)μmol],各组间差异均有显著性意义(P<0.05).结论泽泻活性成分能下调bikunin在结石大鼠肾组织的表达,减少肾组织草酸钙晶体的形成,从而抑制大鼠肾结石形成. 相似文献
3.
OBJECTIVE: To report a simple, relatively rapid protocol to isolate biologically active bikunin from human urine using ion-exchange-trypsin affinity chromatography. Bikunin is a protease inhibitor which has been shown to play a role in various processes, including inhibition of calcium oxalate crystallization, the regulation of proliferation and modulation of carcinogenesis. The unavailability of the purified protein has hampered studies on bikunin's expanding role in these processes. MATERIALS AND METHODS: Female human urine was dialysed (15 kDa threshold) and crudely fractionated with a double-saturated ammonium sulphate precipitation. The first precipitation was with 35% saturated ammonium sulphate, and the supernatant was harvested, and the second with 90% saturated ammonium sulphate, and the precipitate collected. The protein mixture was then passed over Sepharose SP-fast-flow cation exchange and Sepharose Q-fast-flow anion exchange columns connected in series. The final purification was with a trypsin-affinity column which selectively bound bikunin. RESULTS: This procedure could recover 1 microg of bikunin per 2 mL of urine, and the final product was essentially free of contaminating inter-alpha-trypsin inhibitor heavy chains or bikunin-heavy chain conjugates. Product purity was confirmed by two-dimensional polyacrylamide gel electrophoresis combined with silver staining or Western blot. All isolations contained the 17 kDa minimally glycoslyated/sulphated form of bikunin and the 28 kDa form of bikunin. Some preparations also contained 33-48 kDa forms of bikunin. The protein cores of all three proteins were confirmed to be bikunin by mass spectrometry and Western blot. Harvested bikunin retained its trypsin inhibitory activity (L-benzoylarginine-p-nitroanilide assay). Preparations containing the 33-45 kDa form had two to three times more trypsin inhibitory activity than preparations without this band. CONCLUSIONS: This novel ion exchange-trypsin affinity chromatography protocol uses only two chromatographic steps. The product consists of three isomers of biologically active bikunin, free of contaminating heavy chains or bikunin-heavy chain conjugates. The ready availability of purified bikunin should facilitate future studies of bikunin's emerging role in urolithiasis, proliferation and carcinogenesis. 相似文献
4.
UTI是一种对酸对热较稳定的丝氨酸蛋白酶抑制物 ,近年来对其来源、结构、功能以及它与疾病的关系都有较深入的研究 相似文献
5.
SHIZUKA IIDA AMMON B. PECK KAREN J. BYER SAEED R. KHAN 《The Journal of urology》1999,162(4):1480-1486
6.
Bikunin plus paclitaxel markedly reduces tumor burden and ascites in mouse model of ovarian cancer 总被引:5,自引:0,他引:5
Kobayashi H Yagyu T Inagaki K Kondo T Suzuki M Kanayama N Terao T 《International journal of cancer. Journal international du cancer》2004,110(1):134-139
Our previous studies of intraperitoneal ovarian carcinoma in a mouse model demonstrated that bikunin gene transfection could prevent ascites formation and intraperitoneal disseminated metastasis. Although ascites was almost completely inhibited, tumor burden was variably reduced. Several reports have indicated that bikunin may be involved in tumor survival. In the present study, the effectiveness of exogenous bikunin and the biodistribution characteristics of (125)I-bikunin were initially examined in a mouse model of human ovarian cancer HRA cells. The once-daily i.p. administration of bikunin significantly decreased progressive growth of HRA tumors and ascites formation in a dose-dependent manner. Maximal radioisotope tumor uptake peaked at 7.4% injected dose/g at 3 hr. Bikunin binding specificity was demonstrated by reduced tumor uptake after coinjection of excess nonradioactive bikunin. Bikunin was rapidly excreted renally. The bikunin therapy produced the significant inhibition in expression of the proteolysis (uPA and uPAR) and angiogenesis-related molecules (VEGF and bFGF). The second purpose of our study was to optimize the antimetastatic activity of bikunin in combination with paclitaxel against HRA cells growing orthotopically in mice. The once-daily i.p. administration of bikunin (25 microg/g body weight/day) in combination with paclitaxel (i.p., 100 microg/20 g at days 2 and 5) significantly decreased progressive growth of HRA cells in a synergistic fashion. In conclusion, combination therapy with bikunin plus paclitaxel may be an effective way to markedly reduce i.p. tumor growth and ascites in ovarian carcinoma possibly through suppression of uPA, uPAR, VEGF and bFGF expression. 相似文献
7.
目的探讨α-1-微球蛋白/胰蛋白酶抑制剂前体(alpha-1-microglobulin/bikunin precursor,AMBP)在胃癌患者血清中的表达及其在胃癌早期诊断的价值。方法收集北京大学肿瘤医院2007-07-05-2008-12-20收治的114例胃癌患者和49名健康体检者的血清,114例胃癌患者分为胃癌早期组(49例)和胃癌晚期组(65例)。采用蛋白质印迹法和酶联免疫吸附试验(enzyme-linked immunosorbent assay,ELISA)检测血清中AMBP水平,分析其与胃癌临床病理参数的相关性。结果蛋白质印迹法检测结果显示,正常组AMBP血清平均表达量为22.62±2.91,胃癌早期组为32.19±1.53,胃癌晚期组为41.61±3.19,差异有统计学意义,F=12.04,P=0.002 2;组间两两比较差异亦有统计学意义,P值均<0.05。ELISA检测结果显示,正常组AMBP血清平均表达量为(8.156±0.154)μg/mL,低于胃癌患者的(8.948±0.107)μg/mL,t=4.140,P<0.001;在95%可信度下,取阈值为8.546μg/mL时,其诊断敏感性为71.05%,特异性为63.27%;胃癌早期组AMBP血清平均表达量为(8.717±0.173)μg/mL,晚期组为(9.122±0.131)μg/mL,均高于正常组(8.156±0.154)μg/mL,P值均<0.05。AMBP血清表达量与患者年龄(P=0.153 9)、性别(P=0.143 1)、肿瘤大小(P=0.080 9)、淋巴结转移(P=0.073 7)无关,而与肿瘤侵袭程度有相关性,P=0.021 8。结论胃癌患者血清AMBP水平显著升高,可能是胃癌早期诊断潜在肿瘤标志之一。 相似文献
8.
Kazuhiro Yoshimura Osamu Miyake Masao Tsujihata Toshiaki Yoshioka Seiji Yamaguchi Takuo Koide Shiro Takahara Akihiko Okuyama 《International journal of urology》1999,6(11):567-571
BACKGROUND: There are only a few papers reporting on the role of proteoglycan core protein in calcium oxalate stone formation. The present study was carried out to investigate the role of core protein of proteoglycan in human urine on calcium oxalate (CaOx) crystallization. METHODS: Proteoglycans were collected from whole human urine. The covalently bound glycosaminoglycans (GAG) of proteoglycans were then digested by GAG lyase. The inhibitory activity on CaOx crystal growth in vitro was measured before and after enzyme digestion of proteoglycans. Sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the core protein of proteoglycans and the analysis of amino acid sequence were performed. RESULTS: The core protein showed significant inhibitory activity on CaOx crystal growth, which scarcely changed when compared with that of proteoglycans before enzyme digestion. The SDS-PAGE revealed that the core protein was a single unit with a molecular weight of 26 kDa and amino acid sequencing demonstrated high homology to interalpha-trypsin inhibitor (ITI) light chain (bikunin) with Kunitz inhibitor domain as a core protein. CONCLUSIONS: The results suggested that human urine contains proteoglycans and a major part of them is ITI light chain (bikunin). The Kunitz inhibitor domain, a core protein of bikunin, has significant inhibitory activity on CaOx crystallization without GAG bound covalently to the core protein. 相似文献
9.
Identification of bikunin isolated from human urine inhibits calcium oxalate crystal growth and its localization in the kidneys 总被引:1,自引:0,他引:1
MITSUHIKO OKUYAMA SATOSHI YAMAGUCHI SUNAO YACHIKU 《International journal of urology》2003,10(10):530-535
BACKGROUND: We previously reported a high molecular weight substance purified from human urine that strongly inhibited calcium oxalate (CaOx) crystal growth in vitro. In the study present herein, we identified and investigated a protein purified from human urine that strongly inhibits CaOx crystal growth using a column chromatography series. METHODS: The protein was identified by amino acid sequencing and was investigated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and immunohistochemical staining. RESULTS: The molecular weight of this protein was approximately 35 KDa, and it also had another band around 20 KDa. We determined that the amino acid sequence of the protein was homologous with that of bikunin, the light chain of the inter-alpha-trypsin inhibitor, which is known as a strong CaOx crystallization inhibitor in vitro. On western blotting analysis, the molecular weight was also found to be around 35K Da, the same as that of bikunin. Immunohistochemical staining revealed that it was mainly located in the epithelial cells of the proximal tubules and the thin descending segment near the loop of Henle, but not in the glomeruli, distal tubules or the collecting ducts. CONCLUSION: In the present study, the protein extracted from human urine was identical to bikunin, which may be expressed mainly in the proximal tubules and the thin descending segment near the loop of Henle, and which prevents CaOx crystallization in vitro. 相似文献
10.
Enhanced spontaneous metastasis in bikunin-deficient mice 总被引:2,自引:0,他引:2
Yagyu T Kobayashi H Matsuzaki H Wakahara K Kondo T Kurita N Sekino H Inagaki K 《International journal of cancer. Journal international du cancer》2006,118(9):2322-2328
Previously, we showed that bikunin, a Kunitz-type protease inhibitor, inhibits invasion and metastasis in several types of cancer cells possibly through suppression of upregulation of urokinase-type plasminogen activator (uPA) expression. Bikunin corresponds to a light chain of the inter-alpha inhibitor. To explore critical role of endogenous bikunin, we used bikunin knockout (Bik-/-) mice. Here, we show that 1) higher frequency of spontaneous 3LL lung metastasis was observed in Bik-/- mice compared to Bik+/+ mice, suggesting that bikunin deficiency increases the sensitivity of mice to lung metastasis; 2) administration of exogenous bikunin caused a significant reduction of lung metastasis in Bik-/- and Bik+/+ mice; 3) primary and metastatic tumors significantly upregulated uPA and PAI-1 expression in Bik-/- mice relative to Bik+/+ mice at least through phosphorylation of ERK1/2 and 4) exogenous bikunin suppressed phosphorylation of ERK1/2 and upregulation of uPA and PAI-1 expression in 3LL cells in response to G-CSF. These data allow us to conclude that the increased sensitivity of Bik-/- mice to lung metastasis in vivo is due to a lack of circulating proteins of the inter-alpha inhibitor family, especially bikunin. 相似文献